Phosphoramidite synthones for the synthesis of self-neutralizing oligonucleotide compounds

ABSTRACT

Compositions and compounds of nucleoside phosphoramidites and modified oligonucleotides, each comprising one or more charge-neutralizing moieties according to the formula V 
                         
The nucleoside phosphoramidites permit facile attachment of the neutralizing moieties on the backbones of the modified oligonucleotides. The modified oligonucleotides can be used as therapeutic agents (i.e., oligotherapeutics) for the treatment of cancer, autoimmune disorders, genetic diseases, infectious diseases, neurological diseases, inflammatory diseases, metabolic diseases and others.

RELATED APPLICATIONS

The application is a continuation of U.S. application Ser. No.15/527,845, filed May 18, 2017, issuing Jul. 28, 2020 as U.S. Pat. No.10,723,755, which is a 371 of PCT/US2015/061343, filed Nov. 18, 2015,which claims the benefit of priority to U.S. Provisional Application No.62/081,316, filed Nov. 18, 2014, each of which is incorporated herein byreference in its entirety.

BACKGROUND

Nucleic acids are used as therapeutics, for example, in oligotherapy,antisense therapy, siRNA, and RNAi. However, nucleic acids have lowcellular penetration of the nucleic acid.

Antisense based therapies, for example, rely on the hybridization tocomplementary sequences, to allow for the selective silencing ofparticular genes. Thus, oligotherapeutics is a very attractive approachfor the treatment of cancer, genetic mutations, and evenmicroorganism-mediated diseases.

The main hurdle in achieving the potential therapeutic advantages ofthese approaches included poor stability of the oligonucleotides andinefficient intracellular penetration of the oligonucleotides. Thisprompted the development of a large number of synthetic analogs ofnatural oligonucleotides, such as 2′-position modifications,boranophosphonates, locked nucleic acids, peptide nucleic acids (PNA),morpholino derivatives, alkynyl phosphonates, and terminally modifiedoligonucleotides. While these modifications improved the biologicalstability, they did not change the intracellular penetration ofoligonucleotides.

To overcome these challenges, different vehicles have been proposed suchas virus-based delivery systems, liposome formulations, nanoparticles,and transporter chemical groups. For example, tagging the ends of siRNAswith cholesterol, folate, various peptides, and aptamers can aid intransporting oligonucleotides across cellular barriers or in targetingspecific type of cells or organs.

One widely used modification of oligonucleotides is the attachment ofamino groups to oligonucleotides via linkers, mostly used as anchorgroups for post-synthetic derivatization of synthetic oligonucleotides.The linker is usually attached to the 5′-end of the oligonucleotide uponthe completion of automated synthesis. Attachment to the 3′-end onnon-standard supports has also been explored. Attachment of linkers tothe internucleotide phosphates has been performed as well.

However, despite the development of a large number of chemicalmodifications of oligonucleotides including different delivery systems,the absence of available oligonucleotides having effective cellularuptake remains an unmet need. Accordingly, there is a need forcompositions, compounds and systems for modified oligotherapeutics andto enhance the cellular uptake of oligonucleotides for the treatment ofdiseases and disorders.

SUMMARY

The present invention relates generally to compositions and compoundshaving derivatives of synthetic oligonucleotides with one or morecharge-neutralizing moieties on their backbone. The compositions andcompounds described herein can be used as therapeutic agents (i.e.,oligotherapeutics) for the treatment of cancer, autoimmune disorders,genetic diseases, infectious diseases, neurological diseases,inflammatory diseases, metabolic diseases and others.

In some embodiments, the present invention relates to a compound havingstructure (I):

In the compound having structure (I): R₁ is a nucleic acid moiety, aspacer group, or a combination thereof. In some embodiments, R₂ is,independently for each occurrence, selected from the group consisting ofH, CH₃, CH₂CH₃, an alkyl, a substituted alkyl, a branched chain alkyl,formyl, acetyl, CF₃, trifluoroacetyl, allyl, triphenylmethyl, andtert-butyloxycarbonyl. In some embodiments, R₃ is, independently foreach occurrence, selected from the group consisting of H, F, CH₃,CH₂CH₃, an alkyl, a substituted alkyl, a branched chain alkyl, formyl,acetyl, CF₃, trifluoroacetyl, allyl, triphenylmethyl,tert-butyloxycarbonyl, N-PAC, N-iPrPAC, N-benzoyl, and N-Ac.

In some embodiments, each R₃ pair that is bonded to a single nitrogentogether form a ringed-structure (e.g., a 5 membered ring, a 6 memberedring, or a 7 membered ring). Examples of nitrogen-containing ringedstructures include pyrrolidinyl, piperidinyl, piperazidinyl, andmorpholinyl.

In some embodiments, R₄ is selected from the group consisting of H, F,CH₃, CH₂CH₃, OCH₃, OCH₂CH₃, OCH(CH₃)₂, CH(CH₃)₂, and C(CH₃)₃.

In the compound having structure (I), X₁, X₂, and X₃ are, independentlyfor each occurrence, selected from the group consisting of O, S, CH₂,and CH₂CH₂. In some embodiments, m is, independently for eachoccurrence, 0, 1, 2, 3, 4 or 5.

In some embodiments, the compound having structure (I) comprises apharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the nucleic acid moiety has structure (II):

In some embodiments, R₅ in structure (II) is N(CH(CH₃)₂)₂. In someembodiments, R₆ in structure (II) is selected from the group consistingof protected OH, protected SH, protected NH₂, H, OCH₃, OCH₂CH₃, F, Cl,N₃, OCH₂CH₃, OCH₂OCH₂CH₃, SCH₃, and N(CH₃)₂.

In some embodiments, R₇ in structure (II) is a 5′ protecting group and Bis a nitrogenous base.

In some embodiments, the compound having structure (I), each R₃ is H orCH₃, and wherein X₁ is CH₂, X₂ is O, and X₃ is CH₂CH₂.

In some embodiments, the compound having structure (II), the 5′protecting group is selected from the group consisting ofdimethoxytrityl (DMTr), monomethoxytrityl (MMTr), and trityl (Tr).

In some embodiments, the nitrogenous base is a purine or a pyrimidine.For example, a purine can be adenine or guanine; and a pyrimidine can becytosine, thymine, or uracil. In some embodiments, the nitrogenous baseis a modified nitrogenous base. A modified nitrogenous base can include,for example, 5-methylcytosine, pseudouridine, dihydrouridine,7-methylguanosine, hypoxanthine, xanthine, 7-methylguanine,5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine.

In some embodiments, the nitrogenous base comprises a protecting group.Examples of protecting groups include, for example, N-PAC, N-iPrPAC,N-benzoyl, and N-Ac.

In some embodiments, the compound having structure (I) has a spacergroup. For example, the spacer group can be SCH₂, OCH₂, CH₂OCH₂,CH₂SCH₂, CH₂, CH₂CH₂ and CH₂CH₂CH₂. The spacer group can be covalentlybonded between the compound having structure (I) and the nucleic acidmoiety.

In some embodiments, the compounds (e.g., having structure (I), (III)and/or (V)) described herein comprise one or more amino groups (e.g.,amines) that are positively charged. In some embodiments, at least oneof the amino groups is positively charged at a pH of about 6 to about 8.In some embodiments, the at least one of the amino groups is positivelycharged at a pH of about 6.5 to about 7.5. In some embodiments, the atleast one of the amino groups is positively charged at a pH of about 7.0to about 7.5. In one embodiment, one or more amino groups are positivelycharged at a pH of about 7.35 to about 7.45. In some embodiments, eachamino group in the compounds described herein is positively charged. Inone embodiment, each amino group is positively charged at a pH of about6 to about 8. In one embodiment, each amino group is positively chargedat a pH of about 6.5 to about 7.5. In one embodiment, each amino groupis positively charged at a pH of about 7.35 to about 7.45.

In some embodiments, at least one terminal nitrogen in the compoundhaving structure (I) comprises an additional R₃ group. In someembodiments, at least one of the terminal nitrogen is a quaternaryamine. For example, the quaternary amine can be N(CF₃)₃ ⁺, N(CH₃)₃ ⁺,N(CH₂CH₃)₃ ⁺.

In some embodiments, the present invention relates to a compound havingstructure (III):

In the compound having structure (III): R₁ is a nucleic acid moiety, aspacer group, or a combination thereof. In some embodiments, R₂ is,independently for each occurrence, selected from the group consisting ofH, F, CH₃, CH₂CH₃, an alkyl, a substituted alkyl, a branched chainalkyl, formyl, acetyl, CF₃, trifluoroacetyl, allyl, triphenylmethyl,tert-butyloxycarbonyl, N-PAC, N-iPrPAC, N-benzoyl, and N-Ac. In someembodiments, Q is, independently for each occurrence, selected from thegroup consisting of O, S, OCH₂, and CH₂.

In some embodiments, the compound having structure (III) comprises apharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compound having structure (III) has a nucleicacid moiety having structure (II):

In some embodiments, R₅ is N(CH(CH₃)₂)₂. In some embodiments, R₆ isselected from the group consisting of protected OH, protected SH,protected NH₂, H, OCH₃, OCH₂CH₃, F, Cl, N₃, OCH₂OCH₃, OCH₂OCH₂CH₃, SCH₃,and N(CH₃)₂. In some embodiments, R₇ is a 5′ protecting group; and B isa nitrogenous base.

In some embodiments, the compounds described herein can have one or morenucleotides, thereby forming an oligonucleotide. Some or all of the oneor more nucleotides can comprise, for example, a naturally occurringnucleotide and/or a non-naturally occurring nucleotide (e.g., a modifiednucleotide and/or a synthetic nucleotide). Each modified nucleotide canhave a neutralizing moiety and can include nucleoside phosphoramiditesand derivatives thereof.

In some embodiments, the present invention relates to anoligonucleotide. The oligonucleotide can comprise an oligonucleotidesugar-phosphate backbone, a plurality of nitrogenous bases, eachnitrogenous base covalently bonded to a sugar unit of thesugar-phosphate backbone, and at least one neutralizing moietycovalently bonded to a phosphorus of the sugar-phosphate backbone. Asused herein “neutralizing moiety” can include compounds having structure(V):

In the compound having structure (V): R₂ and R₃ are, independently foreach occurrence, selected from the group consisting of H, CH₃, CH₂CH₃,an alkyl, a substituted alkyl, and a branched chain alkyl. In someembodiments, R₄ is selected from the group consisting of H, F, CH₃,CH₂CH₃, OCH₃, OCH₂CH₃, OCH(CH₃)₂, CH(CH₃)₂, and C(CH₃)₃.

In the compound having structure (V), X₁, X₂, and X₃ are, independentlyfor each occurrence, selected from the group consisting of O, S, CH₂,and CH₂CH₂. In some embodiments, m is, independently for eachoccurrence, 0, 1, 2, 3, 4 or 5.

In some embodiments, the oligonucleotides described herein can be asingle stranded oligonucleotide or a double stranded oligonucleotide. Insome embodiments, the oligonucleotide can be anoligodeoxyribonucleotide, an oligoribonucleotide, or a small interferingoligoribonucleotide. In some embodiments, the oligonucleotide comprisesabout 5 to about 500 nitrogenous bases. In some embodiments, theoligonucleotide comprises about 1 to about 500 neutralizing moieties.

Another embodiment of the present invention includes using thecompounds, neutralizing moieties and/or oligonucleotides describedherein to treat a disease or a disorder. A method of treating a diseaseor a disorder in a person in need thereof can comprise administering anoligonucleotide to the person. The oligonucleotide comprises at leastone neutralizing moiety having structure (V) (shown herein) and theoligonucleotide is delivered to a cell and modulates a cellularresponse.

In some embodiments, the disease or disorder is a cancer, an autoimmunedisorder, a genetic disease, an infectious disease, a neurologicaldisease, an inflammatory disease, a metabolic disease or a combinationthereof.

In some embodiments, the compounds and compositions described herein canbe used as a oligotherapy. For example, an oligotherapy can include anantisense therapy, an siRNA therapy, and/or a RNAi therapy.

In some embodiments, method of modulating a cellular response can be inany cell. For example, the cell be an eukaryotic cell and/or aprokaryotic cell. In some embodiments, the methods described hereinallow for modulation of other non-cellular based agents, such as virusesand prions.

In some embodiments, the method comprises administering a compound(e.g., having structures (I), (III), and/or (V)) comprising apharmaceutically acceptable salt, hydrate, or solvate thereof.

Further understanding of the invention can be obtained by reference tothe following detailed description in conjunction with the associateddrawings, which are described briefly below.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1B illustrate hybridized oligonucleotides comprisingneutralizing moieties for the neutralization of negative charges inaccording to the present invention.

FIGS. 2A-2E illustrate other oligonucleotides of the present inventionneutralizing moieties reducing the overall charge.

FIG. 3 illustrates a schematic synthesis to generate neutralizingmoieties, phosphitylating agents and phosphoramidite monomers.

FIGS. 4A-4B illustrate the viability of HEK293 cells after 24 hourincubation with an oligonucleotide containing 0, 1, and 3 neutralizingmoieties (SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; ZT4, ZT5, and ZT6,respectively) at a concentration of 1 μM (FIG. 4A), and 10 μM (FIG. 4B).

FIGS. 5A-5D illustrate phase-contrast images of the morphology of HEK293cells in the presence of 1 μM oligonucleotide. Control cells (FIG. 5A)and cells treated with SEQ ID NO:4 (ZT4; FIG. 5B), SEQ ID NO:5 (ZT5;FIG. 5C), and SEQ ID NO:6 (ZT6; FIG. 5D) are shown at 40×.

FIGS. 6A-6D illustrate phase-contrast images of the morphology of HEK293cells in the presence of 10 μM oligonucleotide. Control cells (FIG. 6A)and cells treated with SEQ ID NO:4 (ZT4; FIG. 6B), SEQ ID NO:5 (ZT5;FIG. 6C), and SEQ ID NO:6 (ZT6; FIG. 6D) are shown at 40×.

FIGS. 7A-7C illustrates the viability of HeLa (FIG. 7A), MCF7 (FIG. 7B),and A172 (FIG. 7C) cells incubated with 10 μM oligonucleotide havingdifferent numbers of neutralizing moieties indicated on the x-axis(corresponding to SEQ ID NOS:11-16; ZT11-ZT16). Cells were counted after96 hrs.

FIGS. 8A-8B illustrates phase-contrast images at 40× of HeLa cellstreated with 10 μM oligonucleotide comprising 0 (SEQ ID NO:15; ZT15)(FIG. 8A) and 4 neutralizing moieties (branched chemical groups) (SEQ IDNO:12; ZT12) (FIG. 8B).

FIGS. 8C-8D illustrates phase-contrast images at 40× of MCF7 cellstreated with 10 μM oligonucleotide comprising 0 (SEQ ID NO:15; ZT15)(FIG. 8C) and 4 neutralizing moieties (branched chemical groups) (SEQ IDNO:12; ZT12) (FIG. 8D).

FIG. 9A illustrates penetration kinetics in MCF cells of variousoligonucleotides (ZT17-ZT20 corresponding to SEQ ID NOS:17-20) withneutralizing moieties having tertiary amino groups at the terminal ends.

FIGS. 9B-9C illustrates fluorescent microscopy images of the uptake ofoligonucleotides having SEQ ID NO:20 (ZT20; FIG. 9B) and SEQ ID NO:17(ZT17; FIG. 9C) in A172 cells after 2.5 hours of incubation.

FIG. 9D illustrates the uptake in A172 cells of various oligonucleotideswith different neutralizing moieties.

FIGS. 10A-10C illustrates the fraction of viable A172 cells using 1, 5,and 10 μM of oligonucleotides that are complementary to miRlOb in A172cells after 24 (FIG. 10A), 48 (FIG. 10B), and 72 hrs. (FIG. 10C) ofincubation.

FIGS. 11A-11I illustrate various embodiments of the compounds (e.g.,oligonucleotides) according to the present invention. FIG. 11A showspart of the structure of SEQ ID NO:23 (ZT23; 13mer), described herein.Two neutralizing moieties, defined by structure (V), neutralize negativecharges on the sugar-phosphate backbone. Each neutralizing moiety has 2terminal quaternary amines (NHCH3)₂+). FIGS. 11B-11G illustrates otherembodiments of oligonucleotides having structures (III) and (II). FIGS.11H-11I illustrates other embodiments of oligonucleotides havingstructures (I) and (II).

DETAILED DESCRIPTION

The present invention relates generally to compositions and compoundshaving derivatives of synthetic oligonucleotides with one or morecharge-neutralizing moieties, and methods of making and using the same.The compositions and compounds described herein can be used astherapeutic agents (i.e., oligotherapeutics) for the treatment ofcancer, autoimmune disorders, genetic diseases, infectious diseases,neurological diseases, inflammatory diseases, metabolic diseases andothers.

The compounds can have one or more nucleotides, thereby formingoligonucleotides.

Some or all of the one or more nucleotides can comprise a naturallyoccurring nucleotide and/or a non-naturally occurring nucleotide (e.g.,a modified nucleotide and/or a synthetic nucleotide).

Each modified nucleotide can have a neutralizing moiety and can includenucleoside phosphoramidites and derivatives thereof. These compounds canhave one or more amino groups that are or can be positively charged. Asdescribed herein, the compounds have chemical structures that promotehybridization (e.g., ion pair) with one or more phosphate groups in anucleic acid molecule The compositions and compounds can neutralize(e.g., self-neutralize) one or more negative charges on a nucleic acidmolecule (e.g, DNA, RNA). The compounds provided herein can also enhancecell membrane penetration and/or cellular uptake. For example, thecompounds described herein can neutralize all or some of the chargefound on oligonucleotides (e.g., nucleic acid molecules). Byneutralizing all or some of the charge, the nucleic acid molecule canenter a cell (e.g., a eukaryotic cell, a prokaryotic cell). Thecompounds can also penetrate through cell membranes, cell walls, and/orcapsids of organisms and pathogens.

The compositions and compounds of the present invention can allow for alow number of charges and/or presence of some degree of hydrophobicityacross a backbone. Also, the compounds described herein can have: theability to maintain natural hybridization properties with a targetnucleic acid sequence (e.g., gene), sufficient water solubility,stability in the presence of a nuclease, low toxicity at a therapeuticconcentration, and/or an ability to silence a target gene by activationof an cellular system (e.g., enzyme) or to block a target gene byselective hybridization.

The known modifications of oligonucleotides described above havesatisfied some but not all of these criteria. Embodiments of the presentinvention address and solve the problem with cellular uptake ofoligonucleotides as described in the main body of this invention.

The compounds and methods provided herein can also be used for thetreatment of diseases and disorders, such as cancer, autoimmunedisorders, genetic diseases, infectious diseases, neurological diseases,inflammatory diseases, metabolic diseases and others.

A schematic of the compositions and compounds described herein areillustrated in FIGS. 1A-1B and 2A-2E. FIGS. 1A-1B illustrate theneutralization of negative charges in a hybridized oligonucleotideaccording to the present invention. The length of neutralizing moiety 10can be optimized so that terminal positive charges can reach andneutralize neighboring (adjacent) negative charges, e.g., from phosphategroups, on the same strand as illustrated in FIG. 1A. The positivecharges on neutralizing moiety 10 can also reach and neutralize negativecharges in a hybridized (opposite) strand as illustrated FIG. 1B.Referring to FIGS. 1A-1B, “Nu” refers to nucleoside or nucleosideanalog, “P” refers to phosphate, “+” refers to a positive charge, and“−”refers to a negative charge. The dotted lines in FIGS. 1A-1B illustratehydrogen bonding between nitrogenous bases.

FIGS. 2A-2E illustrate other embodiments of the compounds of the presentinvention having neutralizing moieties 10, reducing the overall chargeof an oligonucleotide. FIG. 2A illustrates an oligonucleotide with aneutralizing moiety 10 on each phosphate illustrated. Each neutralizingmoiety 10 illustrated in FIG. 2A comprises two branched chemical groups.Each branched chemical group has a single positive charge at a terminalend. As illustrated in FIG. 2A, each neutralizing moiety 10 contributestwo positive charges to the overall structure of the oligonucleotide.FIG. 2B illustrates an oligonucleotide having two neutralizing moieties,each neutralizing moiety having two branched chemical groups. At theterminal end of each branched chemical group is a positive charge. Asdescribed herein, the positive charge can neutralize a neighboringphosphate charge. FIG. 2C illustrates an oligonucleotide similar to FIG.2B, having one neutralizing moiety 10. FIG. 2D illustrates anoligonucleotide having a neutralizing moiety having two branchedchemical groups. One branched chemical group has a single positivecharge and the second branched group has two positive charges. FIG. 2Eillustrates an oligonucleotide having a neutralizing moiety 10 havingtwo branched chemical groups. In FIG. 2E, each branched chemical grouphas two positive charges—a terminal positive charge and an internalpositive charge.

In some embodiments, an oligonucleotide can have a neutralizing moietyon some or all of the phosphates. For example, an oligonucleotide canhave 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more,20 or more, 50 or more, or 100 or more neutralizing moieties.

In some embodiments, a neutralizing moiety can contain at least a singlepositive charge.

For example, a neutralizing moiety (e.g., see FIGS. 1A-2E) can have 2,3, 4, 5, 6, 7, 8, 9, 10, or more positive charges.

In one embodiment, the invention relates to a composition comprising acompound having structure (I):

In some embodiments of the compound having structure (I), R₁ is anucleic acid moiety, a spacer group, or a combination thereof. As usedherein, a “nucleic acid moiety” refers to any compound having a base(e.g. natural or non-natural nitrogenous base), sugar unit, and a 3 or 5valent phosphorus atom. These include naturally occurring nucleic acidmoieties (e.g., nucleotides) and non-naturally occurring nucleic acidmoieties (e.g., synthetic or derivatives of naturally occurring nucleicacid moieties). For example, a nucleic acid moiety can have a phosphate(nucleotide), a phosphoroamidite (e.g., nucleoside 3′-phosphoramidite),a H-phosphonate (e.g., nucleoside 3′-H-phosphonate), or derivativesthereof In some embodiments, the compound having structure (I) and/or(III) has a spacer group.

For example, the spacer group can be SCH₂, OCH₂, CH₂OCH₂, CH₂SCH₂, CH₂,CH₂CH₂ or CH₂CH₂CH₂. The spacer group can be covalently bonded betweenthe compound having structure (I) or (III) and the nucleic acid moiety.

In some embodiments of the compound having structure (I), R₂ is,independently for each occurrence, selected from the group consisting ofH, CH₃, CH₂CH₃, an alkyl, a substituted alkyl, a branched chain alkyl,formyl, acetyl, CF₃, trifluoroacetyl, allyl, triphenylmethyl, andtert-butyloxycarbonyl.

In some embodiments of the compound having structure (I), R₃ is,independently for each occurrence, selected from the group consisting ofH, F, CH₃, CH₂CH₃, an alkyl, a substituted alkyl, a branched chainalkyl, formyl, acetyl, CF₃, trifluoroacetyl, allyl, triphenylmethyl,tert-butyloxycarbonyl, N-PAC, N-iPrPAC, N-benzoyl, and N-Ac.

In other embodiments of the compound having structure (I), each pair ofterminal R₃ groups form a ringed structure with the nitrogen. Forexample, the ringed structure can comprise a 5 membered ring, 6 memberedring, or a 7 membered ring. In each instance, the ringed structurecontains nitrogen. For example, a nitrogen containing ring can be apyrolidine, piperidine, piperazine, or morpholine group (i.e.,pyrrolidinyl, piperidinyl, piperazidinyl, and morpholinyl).

In some embodiments of the compound having structure (I), R₄ is selectedfrom the group consisting of H, F, CH₃, CH₂CH₃, OCH₃, OCH₂CH₃,OCH(CH₃)₂, CH(CH₃)₂, and C(CH₃)₃.

In the compound having structure (I), X₁, X₂, and X₃ are, independentlyfor each occurrence, selected from the group consisting of O, S, CH₂,and CH₂CH₂. In some embodiments, m is, independently for eachoccurrence, 0, 1, 2, 3, 4 or 5.

For example, the compound having structure (I), each R₃ is H or CH₃, andX₁ is CH₂, X₂ is O, and X₃ is CH₂CH₂.

In some embodiments, the compound having structure (I) comprises apharmaceutically acceptable salt, hydrate, or solvate thereof.

In another embodiment, the present invention relates to a compoundhaving structure (III):

In some embodiments of the compound having structure (III), R₁ is anucleic acid moiety, a spacer group, or a combination thereof. In someembodiments, R₂ is, independently for each occurrence, selected from thegroup consisting of H, F, CH₃, CH₂CH₃, an alkyl, a substituted alkyl, abranched chain alkyl, formyl, acetyl, CF₃, trifluoroacetyl, allyl,triphenylmethyl, tert-butyloxycarbonyl, N-PAC, N-iPrPAC, N-benzoyl, andN-Ac. In some embodiments, Q is, independently for each occurrence,selected from the group consisting of O, S, OCH₂, and CH₂.

In some embodiments, the compound having structure (III) comprises apharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compounds (e.g., having structures (I), (III),and/or (V)) described herein comprise one or more amino groups. The oneor more amino groups can be a primary amine, a secondary amine, atertiary amine, a quaternary amine, a cyclic amine, or a combinationthereof. As used herein “amine” and “amino” are used interchangeably torefer to an organic compound containing a basic nitrogen atom.

In some embodiments, the compounds (e.g., having structures (I), (III),and/or (V)) described herein, have 1 or more, 2 or more, 3 or more, 4 ormore, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 ormore amino groups. In one embodiment, the compound has 1 amino group. Inone embodiment, the compound has 2 amino groups. In one embodiment, thecompound has 3 amino groups. In one embodiment, the compound has 4 aminogroups. In one embodiment, the compound has 5 amino groups. In oneembodiment, the compound has 6 amino groups.

In some embodiments, some or all of the amino groups (e.g., in aneutralizing moiety) can be positively charged. For example, 1, 2, 3, 4,5, 6, or more of the amino groups (amines) in a neutralizing moiety arepositively charged. The positive charge can occur in a variety of waysand conditions. For example, the amine can be a quaternary amine, sothat the nitrogen is always charged. In other embodiments, the amine isa protonated primary, secondary, tertiary or cyclic amine. For example,the amine can be protonated under certain conditions, such as pH. Insome embodiments, at least one amino group is positively charged at a pHof about 6 to about 8 in the compound having structure (I), (III),and/or (V). In some embodiments, at least one amino group is positivelycharged at a pH of about 6.5 to about 7.5. In some embodiments, at leastone of the amino group is positively charged at a pH of about 7.0 toabout 7.5. In one embodiment, one or more amino groups arc positivelycharged at a pH of about 7.35 to about 7.45. In some embodiments, each(i.e., all) amino group in the compounds described herein is positivelycharged.

In some embodiments, each terminal nitrogen in the compound, e.g.,having structure (I), comprises an additional R₃ group. In someembodiments, at least one of the terminal amino groups is a quaternaryamine. For example, the quaternary amine can be N(CF₃)₃ ⁺, N(CH₃)₃ ⁺,N(CH₂CH₃)₃ ⁺.

In some embodiments, at least one positive charge (e.g., on an amine) inthe compounds described herein, e.g., having structure (I), (III),and/or (V), can form an ion pair. The ion pair can be intramolecular,e.g., it binds to another part of the same molecule (e.g., nucleic acidmolecule). For example, the structures of the compounds described hereincan allow for a terminal amino group (e.g., that is positively charged)to bind to a phosphate group (e.g., that is negatively charged) that ispart of the same nucleic acid molecule. The structures of the compoundsdescribed herein can also allow for a terminal amino group (e.g., thatis positively charged) to bind to a phosphate group (e.g., that isnegatively charged) that is part of a different nucleic acid molecule.The phosphate group can be an adjacent phosphate group (i.e., directlynext to). The adjacent phosphate group can be at a 5′ end or a 3′ end.

The compounds described herein can self-neutralize charges found withinthe same compound and/or in a different compound (e.g., a hybridizedcompound). For example, the positive charge(s) can neutralize chargefound in a sugar-phosphate backbone of an oligonucleotide compound.

In some embodiments of the compound having structure (I) and/or (III),the nucleic acid moiety has structure (II):

In some embodiments, R₅ in structure (II) is N(CH(CH₃)₂)₂.

In some embodiments, R₆ in structure (II) is selected from the groupconsisting of OH, SH, NH₂, H, OCH₃, OCH₂CH₃, F, Cl, N₃, OCH₂OCH₃,OCH₂OCH₂CH₃, SCH₃, and N(CH₃)₂. It will be readily apparent to one ofordinary skill in the art, that OH, SH and NH₂ groups can be protected.For example, OH can be protected with acetyl, TOM and/or TBDMS. Forexample, SH can be protected with TOM. For example, NH₂ can be protectedwith acetyl and/or trifluoroacetyl.

In some embodiments, R₇ in structure (II) is a 5′ protecting group. Forexample, the 5′ protecting group is selected from the group consistingof dimethoxytrityl (DMTr), monomethoxytrityl (MMTr), and trityl (Tr).

In some embodiments, B in structure (II) is a nitrogenous base. Forexample, the nitrogenous base can be a purine or a pyrimidine. Forexample, a purine can be adenine or guanine; and a pyrimidine can becytosine, thymine, or uracil. In some embodiments, the nitrogenous baseis a modified nitrogenous base.

Examples of a modified nitrogenous base can include 5-methylcytosine,pseudouridine, dihydrouridine, 7-methylguanosine, hypoxanthine,xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and5-hydroxymethylcytosine.

Examples of modified nucleosides include 5-methylcytidine (5mC),pseudouridine (Ψ), 5-methyluridine, 2′ O-methyluridine, 2-thiouridine,N-6 methyladenosine, hypoxanthine, dihydrouridine (D), inosine (I), and7-methylguanosine (m7G). It should be noted that any number of bases inthe oligonucleotides described herein can be substituted with one ormore modified nucleosides (or nitrogenous base). Tt should further beunderstood that combinations of different modifications may be used.

In some embodiments, the nitrogenous base comprises a protecting group.Examples of protecting groups include, for example, N-PAC, N-iPrPAC,N-benzoyl, and N-Ac.

The compounds of the present invention can be used as monomers (e.g., amodified nucleic acid monomer) in the synthesis of oligomers (e.g.,oligonucleotides). In some embodiments, the present invention relates toan oligonucleotide. The oligonucleotide can comprise an oligonucleotidesugar-phosphate backbone, a plurality of nitrogenous bases, eachnitrogenous base covalently bonded to a sugar unit of thesugar-phosphate backbone, and at least one neutralizing moietycovalently bonded to a phosphorus of the sugar-phosphate backbone.

In some embodiments of the compound having structure (I), the nucleicacid moiety comprises a sugar-phosphate backbone. As used herein, a“sugar-phosphate backbone” refers to any backbone structure of anoligonucleotide (e.g., a nucleic acid sequence). For example, the“phosphate” can refer to phosphate (PO₄ ⁻), PSO₃ ⁻, PS₂O₂ ⁻, PO₄D, orPSO₃D. “D” can refer to CHI or a neutralizing moiety having structure(V). The “sugar” can refer to any sugar, such as a pentose (e.g.,ribose, deoxyribose) or modified pentose.

As used herein “neutralizing moiety” can include compounds havingstructure (V):

In some embodiments of the compound having structure (V), R₂ and R₃ are,independently for each occurrence, selected from the group consisting ofH, CH₃, CH₂CH₃, an alkyl, a substituted alkyl, and a branched chainalkyl. In some embodiments, R₄ is selected from the group consisting ofH, F, CH₃, CH₂CH₃, OCH₃, OCH₂CH₃, OCH(CH₃)₂, CH(CH₃)₂, and C(CH₃)₃.

In some embodiments of the compound having structure (V), X₁, X₂, and X₃are, independently for each occurrence, selected from the groupconsisting of O, S, CH₂, and CH₂CH₂. In some embodiments, m is,independently for each occurrence, 0, 1, 2, 3, 4 or 5.

FIGS. 11A-11I illustrate various embodiments of the compounds (e.g.,oligonucleotides) according to the present invention. FIG. 11A showspart of the structure of SEQ ID NO:23 (ZT23; 13mer), described herein.Two neutralizing moieties, defined by structure (V), neutralize negativecharges on the sugar-phosphate backbone. Each neutralizing moiety has 2terminal quaternary amines (NHCH₃)₂ ⁺). FIGS. 11B-11G illustrates otherembodiments of oligonucleotides having structures (III) and (II). FIGS.11H-11I illustrates other embodiments of oligonucleotides havingstructures (I) and (II).

In some embodiments, the oligonucleotides described herein can be asingle stranded (ss) oligonucleotide or a double stranded (ds)oligonucleotide. In some embodiments, the oligonucleotide can be anoligodeoxyribonucleotide, an oligoribonucleotide, a small interferingoligoribonucleotide, or modified oligonucleotides thereof. Theoligonucleotide can be a linear oligonucleotide. The oligonucleotide canbe a circular oligonucleotides.

In some embodiments, the oligonucleotide sequence can vary in length. Insome embodiments, the oligonucleotide can be about 5 bases to about 500(nitrogenous) bases.

In some embodiments, the oligonucleotide can be about 10 to about 300bases; about 15 to about 150 bases; about 20 to about 200 bases; about30 to about 100 bases; about 40 to about 75 bases; or about 50 to about70 bases in length. In some embodiments, the oligonucleotide has 2 ormore, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more,9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more,15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more,21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more,27 or more, 28 or more, 29 or more, 30 or more, 40 or more, 50 or more,100 or more, 200 or more, 300 or more, or 400 or more, up to about 500(contiguous) nitrogenous bases.

In accordance with the teachings of the present invention, the compounds(e.g., oligonucleotides) can target (e.g., bind to, hybridize to) aspecific nucleic acid sequence in a cell.

The portion of the oligonucleotide sequence that is complementary to atarget nucleic acid sequence can also vary in size. In particularembodiments, the portion of each target nucleic acid sequence to whichthe oligonucleotide is complementary can be about 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 81, 82, 83,84, 85, 86, 87 88, 89, 90, 81, 92, 93, 94, 95, 96, 97, 98, 100 or morenucleotides (contiguous nucleotides) in length. In some embodiments,each oligonucleotide sequence can be at least about 70%, 75%, 80%, 85%,90%, 95%, 100%, etc. identical or similar to the portion of each targetnucleic acid sequence. In some embodiments, each oligonucleotidesequence is completely or partially identical or similar to each targetnucleic acid sequence. For example, each oligonucleotide sequence candiffer from perfect complementarity to the portion of the targetsequence by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, etc. nucleotides. In some embodiments, theoligonucleotide sequences is perfectly complementary (100%) across atleast about 5 to about 50 (e.g., about 20) nucleotides of the targetnucleic acid.

In some embodiments, the oligonucleotide comprises about 1 to about 500neutralizing moieties. For example the oligonucleotide has 1 or more, 2or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 ormore, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 ormore, 15 or more, 20 or more, 25 or more, 50 or more, 100 or more, 200or more, or 400 or more neutralizing moieties.

In some embodiments, the oligonucleotides described herein can becovalently bonded to (e.g., conjugated with) another chemical moiety,forming a conjugated complex. For example, a chemical moiety that can bebonded with the oligonucleotides described herein can be any chemicalmoiety, such as, a non-nucleotide chemical moiety, a linear chemicalmoiety, a branched chemical moiety, a cyclic (homo and hetero) chemicalmoiety, an aromatic chemical moiety, a hydrophobic chemical moiety, ahydrophilic chemical moiety, one or more amino acids, a peptide, aprotein, a steroid, a cholesterol, a triglyceride, a fluorochrome, anantibiotic, a vitamin, a sugar, an antibody or fragment thereof, and/orcombinations thereof. It will be readily apparent to one of ordinaryskill in the art to enhance or modulate the function of the compoundsdescribed herein. That is, a purpose of the oligonucleotide can providetargeting to specific target structures (e.g., nucleic acid sequences).A conjugated complex can, for example, increase affinity to those targetstructures. A conjugated complex can facilitate transport acrossbiological structures (e.g., cell membranes, cell walls, etc.). Aconjugated complex can modulate biophysical or biochemical propertiessuch as, for example, bioavailability, hydrophobicity, solubility,and/or stability.

The composition, compounds, and oligonucleotides of the presentinvention can be synthesized that is apparent to one of ordinary skillin the art. For example, unique nucleic acid sequences (e.g,oligonucleotides) can be synthesized using the compounds andneutralizing moieties according to the teachings of the presentinvention. Nucleic acid sequences can target (e.g., hybridize, bind,etc.) a complementary sequence in a cell. The complementary sequence,for example, can be any nucleic acid sequence, such as a DNA sequence orRNA (e.g., mRNA, tRNA, rRNA, etc.) sequence. Complementary sequences canalso comprise a genomic sequence, a gene.

In some embodiments a gene encodes a polypeptide. In some embodiments agene may not encode a polypeptide. A gene may, for example, comprise atemplate for transcription of a functional RNA, i.e., an RNA that has atleast one function other than providing a messenger RNA (mRNA) to betranslated into protein. Examples, include, e.g., long non-coding RNA(e.g., greater than 200 bases in length, e.g., 200-5,000 bases), smallRNA (e.g., small nuclear RNA), transfer RNA, ribosomal RNA, microRNAprecursor, Piwi-interacting RNAs (piRNAs), small nucleolar RNAs(snoRNAs). In some embodiments a small RNA is 25 bases or less, 50 basesor less, 100 bases or less, 200 bases or less in length. In someembodiments a genomic sequence may be suspected of potentiallycomprising a template for transcription of a functional RNA. A geneticmodification may be made in the sequence to determine whether suchgenetic modification alters the phenotype of a cell or animal or affectsproduction of an RNA or protein or alters susceptibility to a disease.

In some embodiments it is of interest to genetically modify a known orsuspected regulatory region, e.g., a known or suspected enhancer regionor a known or suspected promoter region. The effect on expression of oneor more genes in (e.g., within up to about 1, 2, 5, 10, 20, 50, 100, 500kB or within about 1, 2, 5, or 10 MB from the gene) may be assessed.Binding to the regulatory region can modulate the phenotype of a cell oranimal or affects production of an RNA or protein or alterssusceptibility to a disease.

The compositions and compounds of the present invention can be used totreat a disease and/or a disorder. A method of treating a disease or adisorder in a person in need thereof can comprise administering anoligonucleotide to the person, wherein the oligonucleotide comprises atleast one neutralizing moiety having structure (V) (shown herein) andwherein the oligonucleotide is delivered to a cell and modulates acellular response.

In some embodiments, method of modulating a cellular response can be inany cell. For example, the cell be an eukaryotic cell and/or aprokaryotic cell. Eukaryotic cells include, for example, animal cells(e.g., pigs, mice, rats, sheep, cows, dogs, guinea pigs, non-humanprimates, humans), plant cells and fungal cells. Prokaryotic cellsinclude, for example, bacterial cells, archaeal cells. In someembodiments, the methods described herein allow for modulation of othernon-cellular based agents, such as viruses and prions.

In some embodiments, the oligonucleotides can be used as an oligotherapyor any therapeutic that uses one or more nucleic acids. Examples ofoligotherapies can include antisense therapy, siRNA therapy, RNAitherapy. Oligotherapy can be used, for example for genetic disorders orinfections. The oligonucleotides described herein can be synthesized sothat it can bind to, hybridize, or otherwise be complementary to agenetic sequence of a particular gene. In some embodiments, theoligonucleotide binds to and modulates (e.g., inactivates) a mRNAproduced by that gene. Alternatively, in other embodiments, theoligonucleotide can target a splicing site on pre-mRNA or another siteand modify an exon content of an mRNA.

The oligonucleotides described herein can be used in antisense therapiesfor diseases such as cancers (including lung cancer, colorectalcarcinoma, pancreatic carcinoma, malignant glioma and malignantmelanoma), diabetes, Amyotrophic lateral sclerosis (ALS), Duchennemuscular dystrophy and diseases such as asthma, arthritis and pouchitiswith an inflammatory component.

In some embodiments, the disease or disorder is a cancer, an autoimmunedisorder, a genetic disease, an infectious disease, a neurologicaldisease, an inflammatory disease, a metabolic disease or a combinationthereof.

The compounds and compositions described herein, can be used to treatgenetic disorders. For example, a nucleic acid molecule can be modulatedand targeted by the compound.

In some embodiments, the method comprises administering a compound(e.g., having structures (I), (III), and/or (V)) comprising apharmaceutically acceptable salt, hydrate, or solvate thereof

Examples of cancers that can be treated by compounds comprising at leastone neutralizing moiety having structure (V) include, but is not limitedto, blood cancer (leukemia, lymphoma), bone cancer (osteosarcoma,chondrosarcoma), breast cancer (various carcinomas), eye cancer(melanoma, retinoblastoma), gastrointestinal cancer (hepatoma, bladder,colon, esophagial, pancreatic, stomach tumors), kidney, urinary tractand urethral cancer (renal carcinoma, urothelial carcinoma), muscularsystem cancer (rhabdomyosarcoma, fibrosarcoma), nervous system cancer(gliomas, astrocytomas, meningiomas, pituitary tumors, reproductivesystem cancer (testicular, prostate, cervical, endometrial, ovarian),respiratory system cancer (lung cancers, head and neck cancers), skincancer (melanoma, basal cell carcinoma), and other cancers.

Examples of infectious diseases that can be treated by compoundscomprising at least one neutralizing moiety having structure (V) includebut is not limited to, chronic or latent viral infections (HIV, HCV(hepatitis C virus), HSV (herpes simplex virus), HTLV (humanT-lymphotropic virus)), acute viral infections (Influenza, West Nile,Ebola), Bacterial (Mycobacteria spp., Rickettsia spp., Brucella spp),protozoan (Leishmania spp., Chlamydia spp., Plasmodium spp. Malaria) andothers.

Examples of infectious diseases that can be treated by compoundscomprising at least one neutralizing moiety having structure (V) includebut is not limited to, alpha-1 antitrypsin deficiency (AATD),antiphospholipid syndrome (APS), autism, autosomal dominant polycystickidney disease (ADPKD), Crohn's disease, Cystic Fibrosis, Down disease,Duchenne muscular dystrophy (DMD), Factor V Leiden thrombophilia,Gaudier disease, Hemophilia, Huntington's disease, Parkinson's disease,Wilson disease, polycystic kidney disease, Pelizaeus-Merzbacher disease,Vitelly-form Macular Dystrophy 2, and others.

The compositions and compounds can be synthesized by the methodsdisclosed herein and illustrated in FIG. 3 . One of ordinary skill inthe art can readily appreciate different chemical approaches andsynthetic schemes. The following examples, such as the synthesis of thecompositions and compounds, and FIG. 3 are provided for illustrativepurposes only and are in no way intended to limit the scope of thepresent invention.

EXEMPLIFICATION Example 1: Synthesis of1-Bromo-3-(2-Chloroethoxy)Propan-2-Ol (Compound 1 in FIG. 3)

2-Chloroethanol (161 g, 134 ml, 2 mol) was dissolved in 60 ml anhydrousdichloromethane. Boron trifluoride etherate (1 ml) was added on stirringand the resulted solution was cooled, protected from moisture, to −4° C.Epibromohydrin (54.8 g, 34.2 ml, 0.4 mol) was added drop-wise during 35min with stirring and cooling at −7 to −5° C. The clear solution wasstirred for 3 hrs at 0° C. and then left to warm up to room temperature.The progress of the reaction was monitored by TLC (Petroleum ether/Ethylacetate 2:1) and Hanessian's stain. Upon completion, the reactionmixture was evaporated under vacuum (12 mm Hg) in 45° C. bath to obtain82.6 g (95%) of crude product which appeared as yellowish oil. Accordingto ¹H NMR, this material contained 10% (w/w) 2-chloroethanol. Thisproduct was used in the next step without further purification.Analytically pure material was obtained by vacuum distillation. ¹H NMR(300 MHz, CDCl₃) δ: 3.99 (q, J=5.32 Hz, 1H), 3.79 (m, 2H), 3.65 (m, 4H),3.53 (m, 2H), 2.42 (bs, 1H); ³C NMR (75 MHz, CDCl₃) δ: 72.38, 71.43,69.87, 42.85, 34.81.

Example 2: Synthesis of 2-((2-Chloroethoxy)Methyl)Oxirane (Compound 2 inFIG. 3)

Crude 1-bromo-3-(2-chloroethoxy)propan-2-ol (82.6 g, 0.38 moles) wasdissolved in dry tetrahydrofuran (150 ml). The solution was cooled on anice bath. Tertiary potassium butoxide (1M solution in THF, 360 ml, 0.36mol) was added drop-wise within 30 min on ice bath with cooling andstirring. The mixture was stirred for 1 hr on ice cooling and then wasfiltered under vacuum. The precipitate was washed with petroleum ether(2×150 ml). The combined filtrate and washings were evaporated undervacuum to oil. This oil was dissolved in ether (300 ml), and thesolution was extracted with water (2×200 ml), filtered through a cottonplug, and evaporated under vacuum to give 37 g of crude2-((2-chlorocthoxy)methyl)oxirane. This material was further purified byvacuum distillation at 0.1 mm Hg. After small (0.32 g) pre-run of2-chloroethanol, the main fraction was distilled at 36-45° C. to give28.0 g (57%) of pure 2-((2-chloroethoxy)methyl)oxirane. H NMR (300 MHz,CDCl₃) δ: 3.90-3.73 (m, 3H), 3.65 (m, 2H), 3.46 (dd, J₁=5.9 Hz, J₂=11.7Hz, 1H), 3.18 (m, 1H), 2.82 (dd, J₁=4.2 Hz, J₂=5.0 Hz, 1H), 2.64 (dd,J₁=2.7 Hz, J₂=5.0 Hz, 1H); ³C NMR (75 MHz, CDCl₃) δ: 71.79, 71.32,50.69, 43.97, 42.80.

Example 3: Synthesis of 1,3-Bis(2-Chloroethoxy)Propan-2-Ol (Compound 3in FIG. 3)

Boron trifluoride etherate (0.415 ml) was added drop-wise with stirringto a cooled (−15-−10° C.) 2-chloroethanol (79.6 g, 66.0 ml, 0.988 mol).2-((2-Chloroethoxy)methyl)oxirane (27 g, 0.198 mol) was added drop-wiseduring 30 min with stirring at the same temperature. The reactionmixture was kept at 0-5° C. for 3 hrs, and then concentrated undervacuum (12 mm Hg, 45° C. bath) to an oily liquid. This oil was dissolvedin ethyl acetate (150 ml) and the solution was washed with 10% sodiumcarbonate (50 ml), saturated brine (50 ml), and filtered through acotton plug. The solvent was evaporated on rotary evaporator (12 mm Hg,45° C. bath). The residue was subjected to vacuum distillation to give27.7 g (64.6%) distillate at 115-121° C. (0.1 mmHg). The ¹H and ³C NMRsof this distillate revealed that it contains ca. 10% of the isomeric2,3-bis(2-chloroethoxy)propan-1-ol (compound 4). It was purified fromthis isomer by flash chromatography on silica gel. Distillate (5.3) gwas loaded on a silica gel cartridge (Agela Silica-CS, 120 g) and elutedwith a gradient of ethyl acetate/petroleum ether 1:10 (EA/PE, 2 L) toEA/PE 2:1 (3 L). Fractions 5-32 (15 ml each) containing pure productwere combined and evaporated to give 2.27 g of1,3-bis(2-chloroethoxy)propan-2-ol appeared as colorless oil. Fractions33-90 containing impure product were combined and evaporated andsubjected to a second silica gel chromatography under the sameconditions to give additional 0.72 g of pure product. The yield ofpurified product (compound) 3 was 36%. ¹H NMR (300 MHz, CDCl₃) δ: 3.98(m, 1H), 3.76 (m, 4H), 3.68-3.51 (m, 8H), 2.82 (d, J=4.3 Hz, 1H); ³C NMR(75 MHz, CDCl₃) δ: 72.05, 71.38, 69.34, 42.86.

Example 4: Synthesis of 1,3-Bis(2-Azidoethoxy)Propan-2-Ol (Compound 5 inFIG. 3)

1,3-Bis(2-chloroethoxy)propan-2-ol (8.76 g, 0.04 mol of crudedistillate, containing ca. 10% of isomeric2,3-bis(2-chloroethoxy)propan-1-ol), sodium azide (9.97 g, 0.153 mol)and sodium iodide (7.2 g, 0.048 mol) was stirred in 100 mlN,N-dimethylformamide for 16 hrs at 95-100° C. After the cooling, themixture was filtered and the filtrate was evaporated under vacuum. Theresidue was dissolved in dichloromethane (250 ml) washed with water(4×150 ml), and evaporated under vacuum to give 8.5 g, 92% of crudeproduct as a slightly yellow oil, containing ca. 10% of isomeric2,3-bis(2-azidoethoxy)propan-1-ol. This material was purified in twoportions by silica gel chromatography on Agela Silica-CS cartridges, 120g. The elution was carried out with a gradient of ethylacetate/petroleum ether from 1:10 to 1:2. The fractions containing purematerial (TLC, Hanessian's stain) were combined and evaporated, to give,after drying on vacuum and at room temperature (RT), 5.2 g of purecompound 5, as light oil. The mixed fractions were evaporated andsubjected to a second purification under the same conditions asdescribed above to give additional 0.92 g of pure material. Total yieldof pure product (compound) 5 was 6.12 g, 67%. ¹H NMR (300 MHz, CDCl₃) δ:4.00 (m, 1H), 3.72 (m, 4H), 3.66-3.55 (m, 4H), 3.41 (m, 4H), 2.48 (bs,1H); ³C NMR (75 MHz, CDCl³) δ: 72.05, 70.36, 69.31, 50.63.

Example 5: Synthesis of 1,3-Bis(2-Aminoethoxy)Propan-2-Ol (Compound 6 inFIG. 3)

A solution of triphenylphosphine (8.68 g, 33 mmol) in THF (8 ml) wasadded to a solution of compound 5 (3.05 g, 13.2 mmol) in THF (4 ml)under argon with stirring. The flask was equipped with a bubbler andcooled slightly, to maintain the reaction temperature below 30° C. After5 hrs, the release of nitrogen was stopped. Water (0.65 ml) was addedwith stirring, and, again, the flask was cooled slightly to keep thetemperature below 30° C. After 24 hrs, the reaction mixture waspartially concentrated under vacuum (strong foaming was observed), anddiluted with water (100 ml). After 30 min stirring, the whiteprecipitate of triphenylphosphine oxide was filtered under vacuum andwashed with water (3×20 ml). The combined filtrate and washings wereevaporated under vacuum, to give, after drying on high vacuum at r.t.,2.36 g (100%) of product (compound) 6 as a clear oil. ¹H NMR (300 MHz,CDCl₃) δ: 3.99 (m, 1H), 3.54 (m, 8H), 2.89 (t, J=5.2 Hz, 4H), 1.96 (bs,5H). MS (ESI+) m/z: observed, 179.10; calculated for C₇H₁₉N₂O₃ ⁺,179.13.

Example 6: Synthesis of1,3-Bis(2-((Trifluoroacetyl)Amino)Ethoxy)-Propan-2-Ol (Compound 7 inFIG. 3)

Methyl trifluoroacetate (6.77 g, 5.32 ml, 53 mmol) was added drop-wiseto compound 6 (2.36 g, 13.2 mmol) with stirring on ice bath. Theresulted solution was sealed overnight at room temperature. Thevolatiles were evaporated under vacuum, and the residue was purified onsilica gel cartridge (Agela Silica-CS, 120 g) using ethylacetate/petroleum ether 2:1. Fractions containing the product wereevaporated. The residue was dissolved under argon in a mixture ofanhydrous ether and toluene (30 ml of each), and then treated underargon with 30 g molecular sieves 3A in a septum sealed flask. After 3hrs, the solution was removed by a syringe, and the molecular sieveswere washed with 2×20 ml mixture of anh. ether/toluene 1:1. The washingwas conducted under argon using the same syringe technique withoutunsealing the flask. The combined solution and washings were evaporatedunder vacuum to give, after drying under high vacuum at roomtemperature, 3.18 g (65%) of product (compound) 7 as a clear oil, whichwas stored under argon and protected from moisture. ¹H NMR (300 MHz,CDCl₃) δ: 7.03 (bs, 1H), 3.99 (m, 1H), 3.66 (m, 4H), 3.63-3.49 (m, 8H),2.66 (bs, 1H); ³C NMR (75 MHz, CDCl₃) δ: 157.42 (q, J=37.3 Hz), 115.80(q, J=287.2 Hz), 71.95, 69.69, 69.20, 39.64.

Example 7: Synthesis of 1,3-Bis(2-(Dimethylamino)Ethoxy)Propan-2-Ol(Compound 8 in FIG. 3)

1,3-Bis(2-chloroethoxy)propan-2-ol (compound 3, 2.0 g, 9.2 mmol) wasmixed with 24 ml of 40% (w/w) solution of dimethylamine in water,sealed, and stirred for 36 hrs at r.t. The reaction mixture was passedthrough 40 ml of Biorad AG MP-1M anion exchange resin in the OW form.The resin was eluted with water until the eluate became neutral. Theeluates were evaporate under vacuum to give product (compound) 8 (2.3 g,100% yield, contained 6% water) as a white semi-solid substance.Compound 8 was rendered anhydrous by dissolving in a mixture of drytoluene (60 ml), THF (40 ml), and acetonitrile (100 ml), while stirringfor 3 hrs with molecular sieves 4A, filtering anaerobically, washing ofthe molecular sieves with 3×5 ml THF, followed by evaporation of thefiltrate and washings under vacuum. ¹H NMR (300 MHz, D₂O) δ: 3.89 (m,1H), 3.60 (m, 4H), 3.46 (m, 4H), 2.82 (m, 4H), 2.43 (s, 12H); ³¹P NMR(75 MHz, D₂O, ¹H dec.) δ: 71.68, 68.82, 66.39, 56.94, 43.53.

Example 8: Synthesis of5′-O-(4,4′-Dimethoxytrityl)-2′-Deoxytymidine-3′-O—[O-((1,3-Bis(2-((Trifluoroacetyl)Amino)Ethoxy)-2-Propyl)Oxy)-N,N′-Diisopropylphosphoramidite](Compound 12 in FIG. 3)

All procedures were conducted anaerobically under argon using syringe orcannula techniques. The glassware was flame-dried and cooled underargon. All solvents were absolute and septum sealed under argon ornitrogen.

A) Bis(diisopropylamino)chlorophosphine (Compound 9 in FIG. 3)

Diisopropylamine (3.92 g, 5.47 ml, 39 mmol, dried for 3 days undermolecular sieves 3A) and dry toluene (20 ml) were loaded into a roundbottom flask containing a teflon stirring bar. The flask was cooled inice, and phosphorus trichloride (1.22 g, 0.78 ml, 8.88 mmol) was addeddrop-wise with stirring at a rate slow enough to keep the temperature ofthe mixture below 10° C. The flask was equipped with a flame-dried andcooled under argon reflux condenser. The entire content was refluxedwith stirring for 24 hrs. The reaction was controlled by ³¹P NMR inCDCl₃, which showed complete conversion of the PCl₃ singlet at 221.8 ppmto a multiplet at 142.9 ppm.

B)((1,3-Bis(2-((trifluoroacetypamino)ethoxy)-2-propyl)oxy)-N,N,N′,N′-tetraisopropylphosphordiamidite(Compound 10 in FIG. 3)

1,3-Bis(2-((trifluoroacetyeamino)ethoxy)propan-2-ol (7, 2.63 g, 7.10mmol) was dissolved under argon in THF (10 ml) and added drop-wise withstirring to the reaction mixture from step A at −10-−15° C. Afterstirring for 2 hrs at −10° C., the reaction mixture was left to returnto room temperature. The reaction was controlled by ³¹P NMR, whichshowed complete conversion of the multiplet at 142.9 ppm to a multipletat 119.5 ppm. The reaction mixture was filtered by a cannula under argonpressure through a glass fiber filter (the filter was rendered dry byflashing with 50 ml of dry THF under argon). The reaction flask and thefiltered solids were washed with dry THF (3×12 ml). The combinedfiltrate and washings were concentrated under vacuum in 25° C. bath tooil.

C) Compound 12 in FIG. 3 5′-O-(4,4′-Dimethoxytrityl)thymidine (compound11, 2.22 g, 4.08 mmol) was dissolved in 6 ml of dry DMF and evaporatedunder vacuum (35° C. bath). The residue was dissolved under argon in 6ml of dry DMF, and added under argon with stirring to the reactionmixture oil from step B. A solution of 5-ethylthiotetrasole solution inacetonitrile (0.45 M, 3 ml) was added with stirring. After 2 hrs atr.t., ³¹P NMR of the reaction mixture showed conversion of the multipletat 119.5 to two multiplets (representing the two diastereomers ofproduct (compound) 12) at 150.8 and 149.7 ppm, including byproducts at151.4 (multiplet,di-((1,3-bis(2-((trifluoroacetyl)amino)ethoxy)-2-propyl)oxy)-N,N′-tetraisopropylphosphoramidite),and two doublets of multiplets at 17.1 and 7.7 ppm (H-phosphonatehydrolysis byproducts).

Triethylamine (0.5 ml) was added to the reaction mixture, and thevolatilcs were vacuum evaporated. The residue was dissolved indichloromethane containing 1% triethylamine, loaded on a silica gelcartridge (Agela Silica-CS, 120 g), which was pre-equilibrated with 30%ethyl acetate, 1% triethylamine in petroleum ether, and eluted with agradient of 30% ethyl acetate, 1% triethylamine in petroleum ether to 1%triethylamine in ethyl acetate. This resulted in partial purification ofthe product. The final purification was done on a preparative reversephase resin column (100×300 mm). The fractions from the normal phasecolumn containing the product were evaporated, dissolved in 1%triethylamine in methanol (15 ml), loaded on the column, and eluted witha gradient of 50% methanol, 1% triethylamine in water to 1%triethylamine in methanol for 50 min, and then isocratically with 1%triethylamine at methanol for 50 min at a flow rate of 100 ml/min.Fractions containing the product were pooled and evaporated undervacuum. LCMS, and ³¹P NMR analysis of this product showed that itcontained ca. 17% of the corresponding amidate—a byproduct resulted fromoxidation of the product during the reverse phase chromatography. Thismaterial was re-purified by the same chromatographic procedure with thefollowing modifications: the mobile phases were chilled in ice andpurged with helium for 3 hrs before and during the chromatography.Fractions containing compound 12 were pooled and evaporated undervacuum. The residue was evaporated from dry acetonitrile (2×150 ml) andfinally from dry toluene to give 2.44 g (57%) of compound 12 as a whitefoam with 96% purity containing 4% of phosphoroamidate byproduct.Compound 12 consisted of 2 diastereomers in ratio 4:1% (from 1H, ³¹P NMRand HPLC). ¹H NMR (300 MHz, C₆D₆) δ: 7.68, 7.59 (bs, 1H, 6-H), 7.58-6.73(multiple m, 15, DMT Ar—H, CONH), 6.65 (dd, J₁=6.3 Hz, J₂=7.7 Hz, 0.8H,Diast.1 1′), 6.61 (dd, J₁=5.6 Hz, J₂=8.7 Hz, 0.2H, Diast.2 1′), 4.71 (m,1H, H-3′), 4.44, 4.26 (m, 1H, H-4′), 4.11, 4.03 (m, 1H, (OCH₂)₂CHOP),3.61-3.29 (multiple m, 14H, NHCH₂CH₂OCH₂, 5′,5″), 3.334, 3.327 (s, 6H,OCH₃), 3.21, 3.10 (m, 2H, NCHMe₂), 2.35 (m, 2H, H-2′,2″), 1.54, 1.53,1.49 (s, 3H, dT-CH₃), 1.16, 1.12, 1.01 (d, J=6.8 Hz, 12H, NCHCH₃); 3′PNMR (121 MHz, C₆D₆) δ: 150.35 (m, ¹H dec., s), 148.53 (m, ¹H dcc., s).MS (ESI−) m/z: observed, 1042.18 (100.0%), 1043.19 (55.9%), 1044.18(17.6%), 1045.16 (4.9%); calculated for C₄₈H₅₉F₆N₅O₂P [M−H], 1042.39(100.0%), 1043.39 (54.9%), 1044.39 (18.2%), 1045.40 (3.6%). Retentiontimes: Diastereomer 1, 6.44 min; Diastereomer 2, 6.67 min (Column,XBridge C18, 3 2.1×50 mm, mobile phases, A, 10 mM ammonium acetate pH 9,B, acetonitrile, gradient (% B in A) from 0% to 45% for 1 min, then to100% for 5 min, and then isocratic 100% B for 1 min at 0.2 ml/min).

Example 9: Synthesis of5′-O-(4,4′-Dimethoxytrityl)-2′-O-Methyluridine-3′-O—[O-(1,3-Bis(2-((Trifluoroacetyl)Amino)Ethoxy)-2-Propyl)-N,N′-Diisopropylphosphoramidite](Compound 14 in FIG. 3)

Compound 14 was prepared using the procedure for compound 12, with thefollowing modifications: in step A (preparing of compound 9), 5.39 g,7.52 ml, 53 mmol diisopropylamine, 1.22 g, 0.78 ml, 8.88 mmolphosphorous trichloride, and 30 ml toluene were used; in step B(preparing of compound 10), 2.63 g, 7.10 mmol of compound 7 were used;and in step C, 3.28 g, 5.86 mmol of5′-(4,4′-dimetoxytrityl)-2′-O-methyluridine (compound 13) were used. Thepurification was carried out directly on a 100×300 mm reverse phaseresin column using helium de-gased and chilled in ice mobile phases. Theproduct obtained after this purification step contained 17% ofnucleoside (compound) 13, and was re-purified with the same column andprocedure but with extended gradient step—from 50% methanol, 1%tiethylamine in water to 1% triethylamine in methanol for 100 min. Theproduct obtained after this re-purification was 2.76 g (44.4%). Thismaterial had 90% purity (H and ³¹P NMRs, LCMS) and contained 5%nucleoside (compound) 13, and 5% of methylphosphite resulted from areaction of compound 14 with methanol from the mobile phase (replacingthe diisopropylamino group of compound 14 by a methoxy group). Compound14 consisted of 2 diastereomers in ratio 2:1% (from ₁H, ₃₁P NMR andHPLC). ¹H NMR (300 MHz, C₆D₆) δ: 8.10, 7.95 (d, J=8.2 Hz, 1H, H-6),7.62-6.95 (multiple m, 15, DMT Ar—H, CONH), 6.28 (d, J=4.5 Hz, 0.66H,Diast.1′), 6.13 (d, J=2.3 Hz, 0.33H, Diast.2 1′), 5.39, 5.35 (d, J=8.1Hz, 1H, H-5), 4.74, 4.60 (m, 1H, H-2′), 4.50, 4.37 (m, 1H, H-3′), 4.25,4.03 (m, 1H, (OCH₂)₂CHOP), 4.14 (m, 1H, H-4′), 3.70-3.27 (multiple m,14H, NHCH₂CH₂OCH₂, 5′,5″), 3.55, 3.49 (s, 3H, 2′-OMe), 3.377, 3.366,3.347, 3.343 (s, 6H, DTM-OCH₃), 3.19, 3.11 (m, 2H, NCHMe2), 1.15, 1.02(d, J=6.5 Hz, 12H, NCHCH₃); ³¹P NMR (121 MHz, C₆D₆) S 152.98 (m, H dec.,s), 151.54 (m, ¹H dec., s). MS (ESL) m/z: observed, 1058.12 (100%),1059.08 (48.5%), 1060.07 (15.6%), 1061.04 (3.8%); calculated forC₄₈H₅₉F₆N₅O₃P— [M−H], 1058.38 (100.0%), 1059.38 (53.1%), 1060.38(17.5%), 1061.39 (4.1%). Retention times: Diastereomer 1, 4.94 min;Diastereomer 2, 5.23 min (Column, XBridge C18, 3 μm, 2.1×50 mm, mobilephases, A, 10 mM ammonium acetate pH 9, B, acetonitrile, gradient (% Bin A) from 0% to 60% for 1 min, then to 100% for 4 min, and thenisocratic 100% B for 1 min at 0.2 ml/min).

Example 10: Synthesis of5′-O-(4,4′-Dimethoxytrityl)-2′-O-Methyluridine-3′-O—[O-(1,3-Bis(2-((Dimethylamino)Ethoxy)-2-Propyl)-N,N′-Diisopropylphosphoramidite](Compound 16 in FIG. 3)

Compound 16 was prepared using the procedure described for compound 12,with the following modifications: in step A (preparing of compound 9),6.48 g, 9.04 ml, 64 mmol diisopropylamine, 1.47 g, 0.93 ml, 10.7 mmolphosphorous trichloride, and 30 ml toluene were used; in step B(preparing of compound 15), 2.30 g, 8.82 mmol of compound 8 were used;and in step C, 3.28 g, 5.86 mmol of5′-(4,4′-dimetoxytrityl)-2′-O-methyluridine (compound 13) were used. Thepurification was carried out in two runs on a silica gel cartridge(Agela Silica-CS, 120 g) with a gradient of 3% triethylamine in ethylacetate to 20% methanol and 3% triethylamine in ethyl acetate. Fractionscontaining the product were pooled, evaporated under vacuum (25° C.bath), and stripped from residual methanol by re-evaporation from dryacetonitrile containing 1% triethylamine to give, after drying on highvacuum, 3.50 g per run (7.0 g total, 71%) of compound 16 as a foam with92% purity (¹H and ³¹P NMRs, LCMS). It contained 4.5% of thecorresponding H-phosphonate (resulted from hydrolysis of thediisopropylamino group), 3% of the staring nucleoside (compound 13), and1% of the corresponding phosphoroamidite (resulted from oxidation ofcompound 16). Compound 16 was a mixture of 2 diastereomers with ratio53:47% (from ¹H, ³¹P NMR and HPLC). ¹H NMR (300 MHz, C₆D₆): 10.05 (bs,2H, CONH), 8.16, 8.06 (d, J=8.2 Hz, 1H, H-6), 7.69-6.76 (multiple m,12H, DMT Ar—H), 6.20 (d, J=1.8 Hz, 0.47H, Diast.1′), 6.17 (d, J=1.2 Hz,0.53H, Diast.2 5.44, 5.38 (d, J=8.1 Hz, 1H, H-5), 4.89, 4.68 (m, 1H,H-2′), 4.51, 4.45 (m, 1H, H-3′), 4.37-4.21 (m, 1H, (OCH₂)₂CHOP), 4.24,4.16 (m, 1H, H-4′), 3.78-3.45 (multiple m, 14, NHCH₂CH₂OCH₂, 5′,5″),3.65, 3.62 (s, 3H, 2′-OMe), 3.386, 3.369, 3.347, 3.340 (s, 6H,DTM-OCH₃), 2.67-2.47 (m, 2H, NCHMe₂), 2.24, 2.22, 2.19, 2.11 (s, 12H,NMe₂), 1.22, 1.19, 1.10 (d, J=6.7 Hz, 12H, NCHCH₃); ³¹P NMR (121 MHz,C₆D₆) δ: 153.29 (m, ₁H dec., s), 151.47 (m, ₁H dec., s). MS (ESL) m/z:observed, 922.19 (100%), 923.17 (50.7%), 924.12 (16.7%), 925.24 (2.9%);calculated for C₄₈H₆₉N₅O₁₁P— EM−H], 922.47 (100.0%), 923.48 (53.1%),924.48 (16.1%), 925.48 (3.7%). Retention times: Diastereomer 1, 8.41min; Diastereomer 2, 8.85 min (Column, XBridge C18, 3 μm, 2.1×50 mm,mobile phases, A, 10 mM ammonium acetate pH 9, B, acetonitrile, gradient(% B in A) from 0% to 35% for 1 min, then to 100% for 12 min, and thenisocratic 100% B for 1 min at 0.2 ml/min).

Example 11: Synthesis of 1,9-Dichlorononan-5-One (Compound 18 in FIG. 3)from Olean

1,7-Dioxaspiro[5.5]undecane (olean, compound 17, 15 g, 97 mmol,Alfa-Aesar catalog no. B21664) was mixed with conc. HCl (38.7 ml) andthe mixture was heated at 90° C. for 10 min under vigorous stirring. Themixture became quickly homogenous and darkened. After cooling down tor.t., the mixture was extracted with DCM (300 ml), and the DCM extractwas washed with water (150 ml) and 8% sodium bicarbonate in water (150ml), then dried overnight over anhydrous sodium sulfate, filtered andevaporated under vacuum to oil. This oil was subjected to vacuumdistillation (0.1 mm Hg). Three fractions were collected: fraction 1,56-113° C., fraction 2, 113-116° C., and fraction 3, above 116° C.Fraction 2 consisted of pure product (2.3 g, 11.3%). ¹H NMR (300 MHz,CDCl₃) δ: 3.53 (t, J=6.2 Hz, 4H, C1CH₂), 2.45 (t, J=8.8, 4H, COCH₂),1.83-1.66 (m, 8H); ³C NMR (75 MHz, CDCl₃) δ: 209.85, 44.83, 41.89,32.11, 21.20.

Example 12: Synthesis of 1,9-Dichlorononan-5-One (Compound 18 in FIG. 3)from A-Valerolacone

6-Valerolacone (4.00 g, 3.71 ml, 30 mmol) was loaded in a flask equippedwith a reflux condenser, thermometer, and magnetic stirrer. Dry THF (6ml) was added under Ar. The mixture was cooled on ice and NaH (60% inmineral oil, 800 mg, 20 mmol) was added in portions within a couple ofminutes. The cooling bath was removed and the mixture was warmedcarefully with a dryer—exothermic reaction. The heating was turned onand the mixture was bought slowly to reflux. After reflux for 1.5 hrs,the mixture turned into frothing semi-solid state. Additional THF (6 ml)was added. Conc. HCl (8 ml) was added in portions causing a lot offrothing at the beginning. The mixture was reflux briefly, and then thereflux condenser was replaced by a straight one, and ca. 15 ml weredistilled off. Conc. HCl (6 ml) was added again and the distillation wascontinued until the vapor temperature exceeded 100° C. The mixture (twolayers) was cooled and evaporated on rotary evaporator until most of theHCl was evaporated and the salts crystallized. Conc. HCl (8 ml) wasadded once again, the mixture was refluxed vigorously for 20 min andcooled in ice. Salts crystallized again and a couple of milliliters ofwater were added to dissolve them. The mixture was extracted with 2×30ml ether. The ether extract was dried with Na₂SO₄, filtered, and theether was evaporated under vacuum.

The resulting oil was subjected to vacuum distillation (0.1 mm Hg).Three fractions were collected: fraction 1 (45-86° C.), fraction 2(86-160° C.), and fraction 3 (above 160° C.). Fraction 2 (1.4 g)contained the product along with other by-products. Silica gelchromatography of fraction 2 (Agela Silica-CS, 120 g,Ethylacetate/Petroleum Ether, gradient from 1:50 to 1:10) give 249 mg(5.9%) of pure product (compound) 18.

Example 13: Synthesis of 1,9-Dichloro-5-Nonanol (Compound 19 in FIG. 3)

Sodium borohydride (358 mg, 9.5 mmol) was added to ice bath-cooledmixture of 1,9-Dichlorononan-5-one (compound 18, 4.20 g, 20 mmol),methanol (4 ml) and water (2 ml) with vigorous stirring. After 30 min at0-4° C. the reaction mixture was extracted with ethyl acetate (3×50 ml).The extract was dried over Na₂SO₄, filtered, and evaporated under vacuumto give product (compound) 19 (3.92 g, 92%) appeared as a clear oil. HNMR (300 MHz, CDCl₃) δ: 3.63 (m, 1H, CHOH), 3.57 (t, J=6.6 Hz, 4H,ClCH₂), 1.81 (m, 4H, CH₂CH₂Cl), 1.69-1.39 (m, 9H, CH₂, OH); ³C NMR (75MHz, CDCl₃) δ: 71.62, 45.16, 36.81, 32.74, 23.19.

Example 14: Synthesis of 1,9-Bis(Dimethylamino)-5-Nonanol (Compound 20in FIG. 3)

1,9-Dichloro-5-nonanol (compound 19, 3.87 g, 18.8 mmol) was evaporatedunder vacuum from THE (50 ml) to remove any traces of ethyl acetate. Theresidue was mixed with dimethylamine (40% solution in water, 100 ml),sealed, and stirred at r.t. for 20 hrs. The mixture was diluted withwater (200 ml) and extracted with 3×150 ml ether. The ether was removedunder vacuum and the resulted oil was rendered anhydrous by evaporationfrom toluene (2×100 ml) to give 3.3 g of compound 20 (76%) product cameout as clear oil. H NMR (300 MHz, CDCl₃) δ: 3.67 (m, 1H, CHOH), 3.49(bs, 1H, OH), 2.32 (m, 4H, CH₂NMe₂), 2.22 (s, 12H, NMe₂), 1.77-1.46 (m,12, CH₂); ³C NMR (75 MHz, CDCl₃) δ: 71.07, 59.88, 45.50, 38.03, 28.10,23.85.

Example 15: Synthesis of5′-O-(4,4′-Dimethoxytrityl)Thymidine-3′-O—[O-(Bis-(4-(Dimethyamino)Butyl)Methyl)-N,N′-Diisopropylphosphoramidite](Compound 22 in FIG. 3)

Compound 22 was prepared using the procedure described for compound 12,with the following modifications: in step A (preparation of compound 9)4.30 g, 5.99 ml, 42 mmol diisopropylamine, 0.972 g, 0.62 ml, 7.08 mmolphosphorous trichloride, and 20 ml toluene were used; in step B(preparation of compound 21), 1.50 g, 6.51 mmol of compound 20 wereused; and in step C, 3.85 g, 7.08 mmol of5′-(4,4′-dimetoxytrityl)-thymidine (compound 11), and 3 mmol (6.6 ml of0.45 M solution in acetonitrile) of ethylthiotetrazole were used. Also,in step B after filtration, dimethylamine (2 ml) was added, and thencompound 20 was added as neat oil, and the mixture reacted at roomtemperature for 2.5 hrs.

A) Bis(diisopropylamino)chlorophosphine (Compound 9 in FIG. 3 )Diisopropylamine (4.30 g, 5.99 ml, 42 mmol dried over molecular sieves3A) and dry toluene (20 ml) was loaded into a flame dried and cooledunder Ar round bottom flask containing an teflon stirring bar. The flaskwas cooled in ice under Ar and phosphorus trichloride (0.972 g, 0.62 ml,7.08 mmol) was added drop-wise with stirring in ice bath within 1 min.The flask was equipped with a flame-dried and cooled under argon refluxcondenser, and the content was refluxed with vigorous stirring for 20hrs. The reaction was controlled by ³¹P NMR in C₆D₆, which showedcomplete conversion of the PCl₃ singlet at 221.8 ppm to a multiplet at132.3 ppm, and small amount (8%) of doublet of multiplets centered at2.57 ppm, representing a hydrolysis product (H-phosphonate).

B)O-(bis-(4-(dimethyamino)butyl)methyl)-N,N,N′,N′-tetraisopropylphosphordiamidite(Compound 21 in FIG. 3)

1,9-Bis(dimethylamino)-5-nonanol (20) (compound 20, 1.50 g, 6.51 mmol)was dried by vacuum evaporation from dry toluene (2×50 ml). Drydimethylamine (2 ml, 1.43 g, 14 mmol) followed by compound 20 was addedwith stirring under Ar to the cooled (r.t.) reaction mixture from stepA. The mixture was stirred for 2.5 hrs at r.t. and under Ar. Thereaction was controlled by ³¹P NMR in C₆D₆, which showed completeconversion of the multiplet at 132.3 ppm to a multiplet at 107.5 ppm.The reaction mixture was filtered by a cannula under argon pressurethrough a glass fiber filter (the filter was rendered dry by flashingwith 50 ml of dry THE under argon). The reaction flask and the filteredsolids were washed with dry THF (3×12 ml). The combined filtrate andwashings were concentrated under vacuum (25° C. bath) to oil.

C) Compound 22 in FIG. 3 5′-O-(4,4′-Dimethoxytrityl)thymidine (compound11, 3.85 g, 7.08 mmol) was dissolved in 50 ml of dry DMF andconcentrated under vacuum (35° C. bath) to ¼ of its original volume. Theresulted solution was added under argon with stirring to the reactionmixture oil from step B. A solution of 5-ethylthiotetrasole inacetonitrile (0.45 M, 6.6 ml) was added with stirring. After two hrs 15min at r.t., ³¹P NMR of the reaction mixture showed conversion of themultiplet at 107.5 to two multiplets (representing the two diastereomersof compound 22) at 144.7 and 144.1 ppm. Triethylamine (1 ml) was addedto the reaction mixture and the volatiles were evaporated under vacuum.The residue was dissolved in a minimal amount of ethyl acetatecontaining 3% triethylamine, loaded on a silica gel cartridge (AgelaSilica-CS, 120 g), which was pre-equilibrated with the same solvent, andeluted with a gradient of 3% triethylamine in ethyl acetate to 3%triethylamine in ethyl acetate/methanol 4:1. Fractions containing theproduct were pooled and evaporated under vacuum. The residue wasevaporated from toluene (2×150 ml) and finally from dry toluene (50 ml)to give, after drying for 3 hrs at high vacuum, 4.95 g (77%) of compound22 as a white foam with 96% purity containing 2% of phosphoroamiditcbyproduct. Compound 22 consisted of 2 diastereomers with ratio 1:1%(from 1H, ³¹P NMR and HPLC). ¹H NMR (300 MHz, C₆D₆) δ: 7.64-6.74 (mm,14H, 6-H, DMT Ar—H), 6.65 (m, 1H, 1′), 4.77 (m, 11H, H-3′), 4.43, 4.29(two m, 1H, H-4′ of diast.1 and diast.2), 3.94, 3.80 (two m, 1H,(OCH₂)₂CHOP of the two diastereomers), 3.68-3.29 (mm, 4H, 5′,5″,NCHMe₂), 3.35, 3.34 (s, 6H, OCH₃), 2.63-2.50 (mm, 2H, H-2′,2″), 2.40 (t,J=7.2 Hz, 4H, NCH₂), 2.35 (s, 3H, dT-CH₃), 2.26, 2.23, 2.16, 2.11 (s,12H, N(CH₃)₂, 1.76-1.39 (mm, 12H, CH₂), 1.22, 1.19, 1.15, 1.03 (d, J=6.9Hz, 12H, NCHCH₃); ³¹P NMR (121 MHz, C₆D₆) 6:145.15 (m, ¹H dec., s),145.43 (m, ¹H dec., s). MS (ESI⁺) m/z: observed, 903.94 (100.0/%),904.95 (40.9%), 905.90 (12.3%), 906.88 (2.6%); calculated forC₅₀H₇₅N₅O₈P [M+H]⁺, 904.54 (100.0%), 905.54 (55.2%), 906.54 (17.6%),907.55 (2.7%). Retention times: Diastereomer 1, 6.34 min; Diastereomer2, 6.72 min (Column, XBridge C18, 3 μm, 2.1×50 mm, mobile phases, A, 10mM ammonium acetate pH 9, B, acetonitrile, gradient (% B in A) from 0%to 35% for 1 min, then to 100% for 8 min, and then isocratic 100% B for1 min at 0.2 ml/min).

Example 16: Synthesis of Oligonucleotides Containing NeutralizingMoieties at the Target Locations

Compounds 12, 14, 16, and 22 shown in FIG. 3 allowed the incorporationof specific neutralizing moieties in the backbone of an oligonucleotideduring the direct automated synthesis as demonstrated herein.Neutralizing moieties synthesized from compounds 12 and 14 are referredto as compound (i). Neutralizing moieties synthesized from compound 16is referred to as compound (ii). Neutralizing moieties synthesized fromcompound 22 is refined to as compound (iii). Specific structures ofcompounds (i), (ii), and (iii) are illustrated in Table 1 below.

TABLE 1 Structures of Compounds (i), (ii) and (iii) (e.g., neutralizingmoiety) derived from compounds 12, 14, 16, and 22 onto the backbones ofoligonucleotides during automated synthesis Structure of NeutralizingMoiety (NM) Compound ID

Compound (i): 1,3-Bis(2-aminoethoxy)propan-2-ol

Compound (ii): 1,3-Bis(2-(dimethylamino)ethoxy) propan-2-ol

Compound (iii): 9-Bis(dimethylamino)-5-nonanol

Phosporamidite synthones, such as compounds 12, 14, and 16 where usedfor the incorporation of one or more neutralizing moieties with primaryor tertiary amines at the termini into the sugar-phosphate backbones ofthe oligonucleotide. Oligonucleotides were synthesized on a 394 DNA/RNAsynthesizer (Applied Biosystems) using standard phosphoramiditechemistry and mild deprotection phosphoramidite monomers (Glen Research,Sterling, Va. or ChemGenes, Wilmington, Mass.). Coupling rates for newmodified monomers were as good as that for standard phosphoramidites. Asshown, a the variety of oligonucleotides (Table 2) and new monomers werecompatible with phosphoramiditc chemistry.

For instance, the incorporation of FAM labeling groups at the5′-positions of oligonucleotide, 6-Fluorescein Phosphoramidite (FAM)(Cat. #10-1964-90, Glen Research, Sterling, Va.) was used. For theincorporation of thiophosphatc segments, oxidation was performed byusing Beaucage thiolation reagent. Oligonucleotides shown in Table 2were purified on standard C18 HPLC columns by separating compounds with4,4′-Dimethoxytrityl groups from other capped oligonucleotides.Deprotection of synthesized oligonucleotides were performed by 1 hrincubation of controlled porous glass (CPG; a solid support) witholigonucleotide in the mixture of concentrated ammonia: 40% methylamine.MWs of the synthesized oligonucleotide were determined by mass spec (MS)analysis. Excellent correlation between the calculated and MW determinedby mass spectrometry was demonstrated shown in Table 3, below.

TABLE 2 Sequences of Oligonucleotides with Neutralizing Moieties Synthesized and Tested Chemistry: SEQ ID Compound ID (table 1);NO: Sequence (5′-3′) neutralization (%)  1 (ZT1)ATA GTA GTA GTC CTA GTC T DNA—P═O^(a) (i); 16  2 (ZT2)ATA GTA GTA GTC CTA GTC T DNA—P═O (i); 33  3 (ZT3)ATA GTA GTA GTC CTA GTC T DNA—P═O (i); 50  4 (ZT4)UUC GUA GUU GUC UUA GUC C 2′OMe—P═O^(b) (NA); 0  5 (ZT5)UUC GUA GUU GUC UUA GUC C 2′OMe—P═O^(b) (i); 33  6 (ZT6)UUC GUA GUU GUC UUA GUC C 2′OMe—P═O (i); 50  7 (ZT7)UUC GUA GUU GUC UUA GUC C 2′OMe—P═O (i); 100  8 (ZT8)FAM-UUC GUA GUU GUC UUA GUC C 2′OMe—P═O (NA); 0  9 (ZT9)FAM-UUC GUA GUU GUC UUA GUC C 2′OMe—P═O (i); 15 10 (ZT10)FAM-UUC GUA GUU GUC UUA GUC C 2′OMe—P═O (i); 95 11 (ZT11)UCG UAC UUA UCU UAA UCC UAC 2′OMe—P═O (ii); 90 12 (ZT12)UCG UAC UUA UCU UAA UCC UAC 2′OMe—P═O (ii); 60 13 (ZT13)UCG UAC UUA UCU UAA UCC UAC 2′OMe—P═O (ii); 30 14 (ZT14)UCG UAC UUA UCU UAA UCC UAC 2′OMe—P═O (ii); 15 15 (ZT15)UCG UAC UUA UCU UAA UCC UAC 2′OMe—P═O 16 (ZT16)GCG UAG GAU UAA GAU AAG UAC NA; 0 17 (ZT17)FAM-UCG UAC UUA UCU UAA UCC UAC 18 (ZT18)FAM-UCG UAC UUA UCU UAA UCC UAC 2′OMe—P═O (ii); 28 19 (ZT19)FAM-UCG UAC UUA UCU UAA UCC UAC 2′OMe—P═O (ii); 56 20 (ZT20)FAM-UCG UAC UUA UCU UAA UCC UAC 2′OMe—P═O (ii); 86 21 (ZT21)CAC AAA AUC GGU UCU ACA GGG UA 2′OMe—P═S^(c) NA; 0 22 (ZT22)CAC AAA AUC GGU TCU ACA GGG UA 2′OMe—P═S (ii); 55 23 (ZT23)CUG UGG AAG UCU A 2′OMe—P═O (ii); 50 24 (ZT24) CUG UGG AG UCU A2′OMe—P═S NA; 0 25 (ZT25) AGA CTA GGA CTA CTA CTA TT 2′OMe—P═S NA; 026 (ZT26) CAC AAA AUC GGU TCU ACA GGG UA 2′OMe—P═S (ii); 27 27 (ZT27)ATA GTA GTA GTC CTA GTC T 2′OMe—P═O (i); 67 28 (ZT28)FAM-CAC AAA AUC GGU UCU ACA GGG UA 2′OMe—P═O (ii); 55 29 (ZT29)FAM-CAC AAA AUC GGU TCU ACA GGG UA 2′OMe—P═S NA; 0 30 (ZT30)FAM-UUC GUA GUU GUC UUA GUC C 2′OMe—P═O (i); 79 31 (ZT31)ATA GTA GTA GTC CTA GTC T DNA—P═O (NA); 0 ^(a)DNA—P═O indicatesdeoxy-oligonucleotide with phosphate backbones. ^(b)2′OMe—P═O indicates2′OMe derivative of RNA with phosphate backbones. c2′OMe—P═S indicates2′OMe derivative of RNA with thiophosphate backbones. NA = notapplicable. FAM = a fluorescein label.

In the sequences provided in Table 2, above. Underlined bases (e.g., U,T) indicates a location of a neutralizing moiety having the structure ofcompound (i); bases with a double underline (e.g., U, I′) indicates alocation of a neutralizing moiety having the structure of compound (ii).

In Table 2 above, Compound 12 (see FIG. 3 ) was used to synthesize SEQID NOS: 1-3 and 27. Compound 14 was used to synthesize SEQ ID NOS:5-7,9, 10 and 30. Compound 16 was used to synthesize SEQ ID NOS: 11-14,18-23, 26 and 28. SEQ ID NOS:4, 8, 15-17, 24-25, 29 and 31 were used ascontrols.

TABLE 3 Calculated and Actual Molecular Weights determined by massspectrometry (MS). SEQ ID Calc. MW by NO: MW MS 1 (ZT1) 5975.2 5874.7 2(ZT2) 6135.3 6135.6 3 (ZT3) 6295.4 6295.9 4 (ZT4) 6217.1 6217.6 5 (ZT5)6377.7 6377.3 6 (ZT6) 6698.1 6697.3 7 (ZT7) 7177.8 7178.9 8 (ZT8) 6754.26754.6 9 (ZT9) 6914.3 6914.6 10 (ZT10) 7714.9 7715.7 11 (ZT11) 8104.38106.0 12 (ZT12) 7671.9 7672.8 13 (ZT13) 7239.5 7240.6 14 (ZT14) 7023.37024.2 15 (ZT15) 6807.2 6807.9 16 (ZT16) 7075.3 7076.0 17 (ZT17) 7374.37375.4 18 (ZT18) 7806.7 7807.8 19 (ZT19) 8239.0 8240.7 20 (ZT20) 8671.48673.5 21 (ZT21) 8023.9 8026.8 22 (ZT22) 8889.6 8891.5 23 (ZT23) 4945.94946.7 24 (ZT24) 4512.5 4513.6 25 (ZT25) NA 26 (ZT26) NA 27 (ZT27) NA 28(ZT28) NA 29 (ZT29) NA 30 (ZT30) NA 31 (ZT31) NA

Example 16: Preservation of Watson-Crick Hybridization Properties

As it is shown herein in Table 3, melting temperatures (MT) of duplexesregardless of the number of charge neutralizing moieties with primaryamines remain unchanged within the sensitivity of the method (rows 1 to3). Melting was performed for 0.2 μM duplex in buffer with composition:10 mM MgCl₂, 15 mM KCl, 25 mM HEPES, pH 7.3 with heating rate 0.5deg/min. Increasing of the MT was observed for duplexes containing highnumber of charge neutralizing moieties what can be explained by theirhigher hydrophobicity (rows 7 to 10). As it is seen in rows 5 and 11, nomelting was detected when oligonucleotides with BCG (both with primaryand with tertiary amino groups) were mixed with 20-mer 2′OMe scramblesequence. This clearly indicates the absence of non-standardinter-molecular aggregation.

TABLE 4 Melting temperature (MT) of the duplexes containing differentnumber and type of Neutralizing Moiety (NM) # of MT # Duplexes NM (^(°)C.) 1 ZT31:ZT25 0 55.2 2 ZT1:ZT25 1 55.9 3 ZT2:ZT25 2 55.4 4 ZT27:ZT25 456.4 5 ZT27:Scrambled 4 No ON melting 6 ZT15:ZT16 0 56.2 7 ZT14:ZT16 156.5 8 ZT13:ZT16 2 57.5 9 ZT12:ZT16 4 61.2 10 ZT11:ZT16 5 67.2 11 ZT11:Scrambled 5 No ON melting

Example 17: Stability of Oligonucleotides

Stability of some oligonucleotides from the Table 2 was tested at highand low pH (3 to 12) and in serum. During the purification stage,compounds were incubated in concentrated ammonia for over 1 hr and laterin 70% acetic acid for 15 min. All compounds from Table 2 were exposedto a pH ranging from 3 to 12 and didn't decompose. Compounds ZT11 andZT12 were dissolved in PBS and stored at room temperature. HPLC analysisrevealed no decomposition after a month of storage.

For the evaluation of stability of the same compounds in serum, ZT11 andZT12 were dissolved in PBS and each was mixed with ZT15 (the same 2′OMeoligonucleotides with no neutralizing moiety). Both oligonucleotidemixtures were diluted (1:9) with bovine 20 serum. 2 mL reaction mixturewith final 2 o.u. concentrations of oligonucleotide (each) was incubatedat 37° C. Aliquotes of 250 μL were removed at zero, 2, 4, and 8 hrs,diluted with 2 mL of water, and subjected to solid phase extraction withC18 Glen-Pak cartridges (Glen Research). Captured oligonucleotides wereeluted with 20% acetonitrile in water and the eluates were evaporated ona speed-vac. Resulted mixtures of oligonucleotides were analyzed on C18HPLC column and Waters Alliance HPLC system (Waters Corporation).Initially, ratios between ZT15:ZT11 and ZT15:ZT12 were approximately1:1. At the 8 hrs time point, the ratios became 1:12 and 1:10,respectively. The data clearly indicated that incorporation ofneutralizing moieties in the oligonucleotide does not compromise thestability of oligonucleotide and significantly increases their stabilityin nuclease-containing biological fluids.

Example 18: Toxicity

Cytotoxicity of 2′OMe oligonucleotides containing different numbers ofneutralizing moieties (terminated with both primary and tertiary aminogroups) was evaluated in four different cell lines. 2′OMeoligonucleotide without neutralizing moieties were used as controls.

TABLE 5 Oligonucleotides and cell lines used for cytotoxicitydetermination Cell Oligonucleotide tested Observation Line(concentration in μM) time (hrs) HEK293 ZT4, ZTS, ZT6 (1, 10) Up to 24A172 ZT11-ZT15 (1, 5, 10) Up to 96 MCF7 HeLa

Large set of experiments demonstrated that the addition of neutralizingmoieties did not increase the cytotoxicity regardless of their numbers.List of oligonucleotide and cell lines used are shown in Table 4. Effectof oligonucleotide comprising neutralizing moieties was compared to thatof standard 2′OMe oligonucleotides at concentrations of 1 and 10 μM withuntreated cells serving as an additional control. Relative viability ofcells in each test condition was determined as the ratio of propidiumiodide positive (apoptotic cells) to total cell numbers (Hoechst 33342positive cells). Cytotoxicity was also determined visually by assessingbasic cell/monolayer morphology. Viability data for HEK293 cells arepresented in FIGS. 4A-4B, 5A-5D, and 6A-6D.

No visible effects of oligonucleotide on HEK293 were observed during theincubation for up to 24 hrs and for A172, HcLa, and MCF7 cells for up to96 hrs. Average cell viability for HEK293 was 88%-98%, with nosignificant differences in viability between the untreated cells and anyof the oligonucleotide-treated cells (FIGS. 4A-4B).

These results are supported by the visual toxicity data (FIGS. 5A-5D and6A-6D), indicating that the compounds with different degrees of backboneneutralization with neutralizing moieties containing primary aminogroups at the termini are not toxic to cells at concentration as high as10 μM. Similarly, average cell viability for A172, MCF7, and HeLa cellswas over 95% with no significant differences in viability between theuntreated cells and the cells treated with neutralizing moietycontaining oligonucleotides. Viability data for A172, HeLa, and MCF7cells are illustrated in FIGS. 7A-7C and 8A-8B. These data confirmedthat oligonucleotide with backbone neutralized from 14% to as high as80% (ZT11) with neutralizing moieties containing tertiary amino groupsat the termini are not toxic to cells at concentrations as high as 10μM. Referring to FIG. 7A-7C: “Cont” stands for control i.e.no-oligonucleotide-treatment; * indicates a second control using anoligonucleotide with no neutralizing moieties.

Example 19: Effect of Charge Neutralizing Moiety Modifications onCellular Uptake

The effectiveness of the delivery of oligonucleotide comprisingneutralizing moieties into the cytosol of cultured anchorage-dependentcells was studied with three cell lines. All three were developed fromhuman tumors and represent epithelial (HeLa and MCF7) and mesenchymal(A172) cells. All oligonucleotides were labeled with 6-FAM(6-carboxyfluorescein) at 5′-terminus. Cells were grown in DMEM/10% FBS(no antibiotics) were plated on 24- or 96-well plates at a dilutionwhich allows formation of a near-complete monolayer at 18-24 hrs afterthe plating. Alternatively, cells were cultured on glass cover slipsplaced in 60-mm dishes. Test samples dissolved in DMEM/0.5% FBS wereadded and cells were incubated at 37° C. in a CO2 incubator for a fixedperiod of time. At the end of incubation, cells were washed severaltimes, fixed or lysed where necessary, and the amount of cell-associatedfluorescence was measured using a plate reader or fluorescentmicroscope.

Oligonucleotide at a final concentration of 1 μM was added to nearconfluent cells without any formulation or cellular uptake enhancers.Kinetics were evaluated at 30, 60, 120, and 180 minute time points. Asshown in FIG. 9A, significant increase of cellular penetration (3-4times) was demonstrated for oligonucleotide comprising neutralizingmoieties, and this increase was proportional to the number ofneutralizing moieties in the oligonucleotide. For the visualization ofcellular penetration, fluorescent images of A172 glioma cells treatedwith ZT17 (FIG. 9C) and ZT20 (FIG. 9B) for 150 min. and counterstainedwith DAPI. Bright spots correspond to the oligonucleotides labeled with6-carboxyfluorescein (FAM).

Hydrophobicity added by introducing neutralizing moieties also playsvery important role in the enhancement of cellular uptake (FIG. 9D).Neutralizing moieties with tertiary amino groups at the termini (seeFIGS. 2A-2E for structures) provide even higher penetration rate thanneutralizing moieties with primary amino groups. As illustrated in FIG.9D, oligonucleotide (ZT28) with four neutralizing moieties comprisingtertiary amino groups penetrates more efficiently than anoligonucleotide with 5 neutralizing moieties (ZT30) containing primaryamino groups. The poor kinetics of penetration in controloligonucleotides ZT29 and ZT8 (i.e., oligonucleotides withoutneutralizing moieties) are also illustrated.

Example 20: Inhibition of Cell Growth by Targeting MIR10B in A172 GliomaCells

The dose-dependent effect of oligonucleotide on A172 cells wereinvestigated with thiophosphonate oligonucleotide, ZT21 (control,contains no neutralizing moieties), ZT22 (contains 4 neutralizingmoieties, 55% negative charge reduction), and ZT26 (contains 2neutralizing moieties, 27% negative charge reduction) complementary tomiRlOb. miRlOb is heavily presented in the glioblastoma cells and playcrucial role in their uncontrolled proliferation. Cells were seeded in24-well plates and, after overnight incubation, regular growth mediumwas replaced with DMEM/5% FBS supplemented with oligonucleotide atconcentrations of 1, 5, and 101.1 M. Cell behavior was observed dailyusing phase contrast microscopy. Dose-dependent effect on cell growth isillustrated in FIGS. 10A-10C. The labels correspond to theoligonucleotide tested: ZT21 (with no neutralizing moieties), ZT26 (with2 neutralizing moieties), and ZT22 (with 4 neutralizing moieties). Closeto quantitative inhibition of the cell growth was observed for ZT22oligonucleotide at concentration as low as 1 μM. No effect of ZT22 onmiROb-independent HeLa cells was detected.

Example 21: Solubility

Solubility of numerous oligonucleotides from Table 1 in PBS wereevaluated by measuring the UV absorption of the solutions at differentconcentrations of several compounds. All compounds, including highlyneutralized ones (ZT7, ZT1, and ZT27 from Table 1), were completelysoluble at up to 1 mM concentrations.

Those having ordinary skill in the art will appreciate that variouschanges can be made to the above exemplary embodiments without departingfrom the scope of the invention.

What is claimed:
 1. A compound having structure (III):

wherein R₁ is a nucleic acid moiety, optionally connected through aspacer group selected from the group consisting of CH₂OCH₂, CH₂SCH₂,CH₂, CH₂CH₂ and CH₂CH₂CH₂; the nucleic acid moiety having structure (II)

wherein R₅ is N(CH(CH₃)₂)₂, wherein R₆ is selected from the groupconsisting of protected OH, protected SH, protected NH₂, H, OCH₃,OCH₂CH₃, F, Cl, N₃, OCH₂OCH₃, OCH₂OCH₂CH₃, SCH₃, and N(CH₃)₂, wherein R₇is a 5′ protecting group selected from the group consisting ofdimethoxytrityl (DMTr), monomethoxytrityl (MMTr), and trityl (Tr), andwherein B is a 9-purinyl or a 1-pyrimidinyl nitrogenous base, whereinamino groups are protected with a protecting group; wherein R₂ is,independently for each occurrence, selected from the group consisting ofCH₃, CH₂CH₃, an alkyl, a branched chain alkyl, formyl, acetyl, CF₃,trifluoroacetyl, allyl, triphenylmethyl, tert-butyloxycarbonyl,phenoxyacetyl, (4-isopropyl-phenoxy)acetyl, and benzoyl; and wherein Qis, independently for each occurrence, selected from the groupconsisting of O, S, OCH₂, and CH₂; or a pharmaceutically acceptable saltthereof.
 2. The compound of claim 1, wherein each R₂ is CH₃.
 3. Thecompound of claim 1, wherein the 5′ protecting group is dimethoxytrityl(DMTr).
 4. The compound of claim 1, wherein the 9-purinyl or1-pyrimidinyl nitrogenous base is selected from the group consisting of9-adeninyl, 9-guaninyl, 1-cytosinyl, 1-thyminyl, 1-uracilyl,5-methyl-l-cytosinyl, 7-methyl-9-guaninyl, 5,6-dihydro-l-uracilyl, and 5hydroxymethyl-l-cytosinyl, and wherein amino groups of the nitrogenousbase are protected with a protecting group.
 5. The compound of claim 1,wherein the nitrogenous base comprises a protecting group, wherein theprotecting group is selected from the group consisting of phenoxyacetyl,(4-isopropyl-phenoxy)acetyl, benzoyl, and acetyl.
 6. An oligonucleotidecomprising: from 5 to 500 nucleotides; and at least one neutralizingmoiety covalently bonded to a phosphate of the oligonucleotidesugar-phosphate backbone, optionally through a spacer group selectedfrom the group consisting of CH₂OCH₂, CH₂SCH₂, CH₂, CH₂CH₂ andCH₂CH₂CH₂; wherein the at least one neutralizing moiety has structure(III):

wherein R₁ is the point of attachment to the oligonucleotide or spacergroup; R₂ is, independently for each occurrence, selected from the groupconsisting of CH₃, CH₂CH₃, an alkyl, a branched chain alkyl, formyl,acetyl, CF₃, trifluoroacetyl, allyl, triphenylmethyl,tert-butyloxycarbonyl, phenoxyacetyl, (4-isopropyl-phenoxy)acetyl, andbenzoyl; and Q is, independently for each occurrence, selected from thegroup consisting of O, S, OCH₂, and CH₂.
 7. The oligonucleotide of claim6, wherein the oligonucleotide is a single stranded or a double strandedoligonucleotide.
 8. The oligonucleotide of claim 7, wherein theoligonucleotide is an oligodeoxyribonucleotide or anoligoribonucleotide.
 9. The oligonucleotide of claim 6, wherein theoligonucleotide comprises about 1 to about 500 neutralizing moieties.10. The oligonucleotide of claim 7, wherein the oligonucleotidecomprises one or more modified nucleotides and/or synthetic nucleotidescomprising one or more of: (i) a modified nitrogeneous base selectedfrom the group consisting of 5-methylcytosine, dihydrouridine,7-methylguanosine, 7-methylguanine, 5,6 dihydrouracil5,6-dihydro-l-uracilyl, and 5 hydroxymethyl-l-cytosinyl; (ii) a modifiedpentose sugar selected from the group consisting of 2′-OCH₃ ribose and2′-fluoro deoxyribose; and (iii) a modified phosphate selected from thegroup consisting of PSO₃″, PS₂O₂″, PO₄CH₃ and PSO₃CH₃.